Abstract: The potential risk of the spread of COVID-19 epidemic in hospitals poses great threat to the health of medical workers and patients. Hence, it is of great importance to establish a three-level nosocomial infection prevention and control system, improve related measures, and ensure its implementation at each level, so as to effectively prevent and control nosocomial infection. During the prevention and control of the COVID-19 epidemic, medical rescue treatment was jointly performed by local medical workers and various aid teams from other provinces in the designated hospitals in Wuhan, China. A three-level nosocomial infection prevention and control system was established. The highest level consisted of the leaders of original hospitals, the leaders of aid teams, and the persons responsible for nosocomial infection from nosocomial infection management departments and each medical team; the second level was nosocomial infection management department consisting of full-time personnel engaged in nosocomial infection from the original hospital and the medical teams; the third level was the nosocomial infection control group of each ward, including the department leader and the head nurse. This system enabled the medical workers to conduct the prevention and control work in an orderly manner and effectively reduced the incidence rate of nosocomial infection in designated hospitals.